Separation of polynucleotide mixtures is a focus of scientific interest, and numerous researchers have been attempting to achieve technical improvements in various aspects of polynucleotide separation. Anion exchange separation and reverse phase ion pair chromatography are among the most frequently used methods for separating polynucleotide mixtures.
Samples containing mixtures of polynucleotides can result from total synthesis of polynucleotides, cleavage of DNA with restriction endonucleases or RNA, as well as polynucleotide samples which have been multiplied or amplified using polymerase chain reaction (PCR) techniques or other amplifying techniques.
Previous work has focused on developing rapid, high resolution separations, developing separations based on the size of the polynucleotide fragment rather than the base sequence of the fragment, and on developing the ability to collect separated pure fractions of polynucleotides.
W. Bloch (European patent publication No. EP 0 507 591 A2) demonstrated that, to a certain extent, length-relevant separation of polynucleotide fragments was possible on nonporous anion exchanger separation media using eluting solvents containing tetramethylammonium chloride (TMAC). Y. Ohimya et al. (Anal. Biochem., 189:126-130 (1990)) disclosed a method for separating polynucleotide fragments on anion exchange material carrying trimethylammonium groups. Anion exchangers with diethylaminoethyl groups were used by Y. Kato et al. to separate polynucleotide fragments (J. Chromatogr., 478:264 (1989)).
U.S. Pat. No. 5,585,236 (1996) to Bonn et al. describes a method for separating polynucleotides using what was characterized as reverse phase ion pair chromatography (RPIPC) utilizing columns filled with non-polar, nonporous polymeric beads. High resolution, rapid separations were achieved using an ion pairing agent (triethylammonium acetate), and acetonitrile/water eluting solvent gradient. This work is important because it is the first example of a size dependent, sequence independent chromatographic separation of double-stranded polynucleotides by Matched Ion Polynucleotide Chromatography (MIPC). Such separations are comparable to those effected by gel electrophoresis, which is currently the technology most widely used for polynucleotide separations. Bonn's work makes it possible to automate separations of polynucleotides based on their size alone. This method differs from traditional reverse phase processes. Therefore, the term Matched Ion Polynucleotide Chromatography (MIPC) has been applied to the Bonn process to distinguish it from previously known reverse phase processes.
The invention of parent application Ser. No. 08/748,376 is based on the discovery that trace levels of multivalent metal ions, even when present below the limits of detection, interfere with the MIPC separation process. Special steps to prevent, remove or complex any trace multivalent ions result in enhanced separation of polynucleotides and lower the detection threshold. The inventions of provisional applications Ser. No. 60/049,123 filed Jun. 10, 1997; and Ser. No. 60/063,835 filed Oct. 30, 1997 under 35 U.S.C. .sctn.111(b) are based on the discovery that nitric acid passivated stainless steel, titanium, and PEEK (polyetherether ketone) surfaces were, contrary to popular belief, sources of multivalent metal ion contamination in the MIPC process. The deleterious effect of multivalent metal cations on polynucleotide separations as observed herein has not been previously reported. We believe that all chromatographic processes which are capable of separating polynucleotides on non-polar, wide pore separation media are impaired by the interference of multivalent metal ions.